Human urotensin II

ABSTRACT

Human Urotensin II polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing Human Urotensin II polypeptides and polynucleotides in therapy, and diagnostic assays for such.

This application is a division of application Ser. No. 09/027,381, filedFeb. 20, 1998 now U.S. Pat. No. 6,075,137 which claims the benefit ofU.S. Provisional Application No. 60/072,383, filed Jan. 9, 1998 and U.S.Provisional Application No. 06/073,616, filed Feb. 4, 1998, both ofwhose contents are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides, to their use in therapy andin identifying compounds which may be agonists, antagonists and/orinhibitors which are potentially useful in therapy, and to production ofsuch polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

The drug discovery process is currently undergoing a fundamentalrevolution as it embraces ‘functional genomics’, that is, highthroughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on ‘positional cloning’. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available. There is a continuingneed to identify and characterise further genes and their relatedpolypeptides/proteins, as targets for drug discovery.

SUMMARY OF THE INVENTION

The present invention relates to Human Urotensin II, in particular HumanUrotensin II polypeptides and Human Urotensin II polynucleotides,recombinant materials and methods for their production. In anotheraspect, the invention relates to methods for using such polypeptides andpolynucleotides, including the treatment of acute heart failure;hypotension; hypertension; angina pectoris; myocardial infarction;ulcers; psychotic and neurological disorders, including anxiety,schizophrenia, manic depression, delirium, dementia, severe mentalretardation and dyskinesias, hereinafter referred to as “the Diseases”,amongst others. In a further aspect, the invention relates to methodsfor identifying agonists and antagonists/inhibitors using the materialsprovided by the invention, and treating conditions associated with HumanUrotensin II imbalance with the identified compounds. In a still furtheraspect, the invention relates to diagnostic assays for detectingdiseases associated with inappropriate Human Urotensin II activity orlevels.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to Human Urotensin IIpolypeptides. Such peptides include isolated polypetides comprising anamino acid sequence which has at least 70% identity, preferably at least80% identity, more preferably at least 90% identity, yet more preferablyat least 95% identity, most preferably at least 97-99% identity, to thatof SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptidesinclude those comprising the amino acid of SEQ ID NO:2.

Further peptides of the present invention include isolated polypeptidesin which the amino acid sequence has at least 70% identity, preferablyat least 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, most preferably at least 97-99%identity, to the amino acid sequence of SEQ ID NO:2 over the entirelength of SEQ ID NO:2. Such polypeptides include the polypeptide of SEQID NO:2.

Further peptides of the present invention include isolated polypeptidesencoded by a polynucleotide comprising the sequence contained in SEQ IDNO:1.

Polypeptides of the present invention are believed to be members of theUrotensin II family of polypeptides. They are therefore of interestbecause Urotensin II has been reported from fish and frog sources butthe human gene has not been described. Urotensin II has been reported toboth contract rat aorta strips (Itoh, et al., Br J. Phamacol 104:847-852 (1991)), and to produce potent endothelium-dependent relaxationsand endothelium-independent contractions of rat aorta (Br-J-Pharmacol.May; 91(1): 205-12 (1987)): Urotensin II has a hypertensive effect introut (Le-Mevel, et al., Amer J. Physiol. 271: 1335-43 (1996)) anddogfish (Hazon et al., Amer J. Physiol. 265: 573-576 (1993)), and aspasmogenic effect on amphibian and teleost smooth muscle (Yano, et al.,General Comparative Endocrin. 97: 103-110 (1995)). Urotensin II isdescribed as a neurohormone which is important in the freshwateradaptation of teleosts by increasing the net chloride absorption anddecreasing chloride backflux in intestinal epithelium (Loretz-CA,J-Exp-Zool-Suppl. 4: 31-6 (1990); and Baldisserotto, et al.,Braz-J-Med-Biol-Res. 30: 35-39 (1997)). Furthermore, fish Urotensin IIhas recently been discovered to be the ligand for GPR14 receptor (U.S.patent Ser. No. 08/789,354). Here, we report a potential urotensin IIhomologue gene These properties are hereinafter referred to as “HumanUrotensin II activity” or “Human Urotensin II polypeptide activity” or“biological activity of Human Urotensin II”. Also included amongst theseactivities are antigenic and immunogenic activities of said HumanUrotensin II polypeptides, in particular the antigenic and immunogenicactivities of the polypeptide of SEQ ID NO:2. Preferably, a polypeptideof the present invention exhibits at least one biological activity ofHuman Urotensin II.

The polypeptides of the present invention may be in the form of the“mature” protein or may be a part of a larger protein such as a fusionprotein. It is often advantageous to include an additional amino acidsequence which contains secretory or leader sequences, pro-sequences,sequences which aid in purification such as multiple histidine residues,or an additional sequence for stability during recombinant production.

The present invention also includes include variants of theaforementioned polypetides, that is polypeptides that vary from thereferents by conservative amino acid substitutions, whereby a residue issubstituted by another with like characteristics. Typical suchsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg; or aromatic residues Phe and Tyr. Particularlypreferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 aminoacids are substituted, deleted, or added in any combination.

Polypeptides of the present invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

Based on the processing of fish urotensin II, it is predicted that theHuman urotensin II precursor protein will be processed to an activepeptide having an amino acid sequence consisting of ETPDCFWKYCV (SEQ IDNO: 5). Thus, the Human Urotensin II polypeptides of the presentinvention also relates to mature Human Urotensin II of SEQ ID NO: 5.

In a further aspect, the present invention relates to Human Urotensin IIpolynucleotides. Such polynucleotides include isolated polynucleotidescomprising a nucleotide sequence encoding a polypeptide which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, to theamino acid sequence of SEQ ID NO:2, over the entire length of SEQ IDNO:2. In this regard, polypeptides which have at least 97% identity arehighly preferred, whilst those with at least 98-99% identity are morehighly preferred, and those with at least 99% identity are most highlypreferred. Such polynucleotides include a polynucleotide comprising thenucleotide sequence contained in SEQ ID NO:1 encoding the polypeptide ofSEQ ID NO:2.

Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence that has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, to a nucleotidesequence encoding a polypeptide of SEQ ID NO:2, over the entire codingregion. In this regard, polynucleotides which have at least 97% identityare highly preferred, whilst those with at least 98-99% identity aremore highly preferred, and those with at least 99% identity are mosthighly preferred.

Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence which has at least 70%identity, preferably at least some 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, to SEQ IDNO:1 over the entire length of SEQ ID NO:1. In this regard,polynucleotides which have at least 97% identity are highly preferred,whilst those with at least 98-99% identity are more highly preferred,and those with at least 99% identity are most highly preferred. Suchpolynucleotides include a polynucleotide comprising the polynucleotideof SEQ ID NO:1 as well as the polynucleotide of SEQ ID NO:1.

The invention also provides polynucleotides which are complementary toall the above described polynucleotides.

The nucleotide sequence of SEQ ID NO:1 shows homology with both an EST(Accession number: AA535545) and with a genomic clone (Accession number:Z98884). Furthermore, the nucleotide sequence of SEQ ID NO 1 showshomology with Carp Urotensin II-alpha (J. Neuroscience 6, 2730-2735,1986). The neucleotide sequence of SEQ ID NO:1 is a cDNA sequence andcomprises a polypeptide encoding sequence (nucleotide 118 to 534)encoding a polypeptide of 139 amino acids, the polypeptide of SEQ IDNO:2. The nucleotide sequence encoding the polypeptide of SEQ ID NO:2may be identical to the polypeptide encoding sequence contained in SEQID NO:1 or it may be a sequence other than the one contained in SEQ IDNO:1, which, as a result of the redundancy (degeneracy) of the geneticcode, also encodes the polypeptide of SEQ ID NO:2. The polypeptide ofSEQ ID NO:2 is structurally related to other proteins of the UrotensinII family, having homology and/or structural similarity with GobyUrotensin II (PNAS USA 77(8), 5021-5024, 1980).

Preferred polypeptides and polynucleotides of the present invention areexpected to have, inter alia, similar biological functions/properties totheir homologous polypeptides and polynucleotides. Furthermore,preferred polypeptides and polynucleotides of the present invention haveat least one Human Urotensin II activity.

The present invention also relates to partial or other polynucleotideand polypeptide sequences which were first identified prior to thedetermination of the corresponding full length sequences of SEQ ID NO:1and SEQ ID NO:2.

Accordingly, in a further aspect, the present invention provides for anisolated polynucleotide comprising:

(a) a nucleotide sequence which has at least 70% identity, preferably atleast 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, even more preferably at least 97-99%identity to SEQ ID NO:3 over the entire length of SEQ ID NO:3;

(b) a nucleotide sequence which has at least 70% identity, preferably atleast 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, even more preferably at least 97-99%identity, to SEQ ID NO:1 over the entire length of SEQ ID NO:3;

(c) the polynucleotide of SEQ ID NO:3; or

(d) a nucleotide sequence encoding a polypeptide which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% identity, to the amino acid sequence of SEQID NO:4, over the entire length of SEQ ID NO:4;

as well as the polynucleotide of SEQ ID NO:3.

The present invention further provides for a polypeptide which:

(a) comprises an amino acid sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ IDNO:4;

(b) has an amino acid sequence which is at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to the amino acid sequence of SEQ ID NO:2 over theentire length of SEQ ID NO:4;

(c) comprises the amino acid of SEQ ID NO:4; and

(d) is the polypeptide of SEQ ID NO:4;

as well as polypeptides encoded by a polynucleotide comprising thesequence contained in SEQ ID NO:3.

The nucleotide sequence of SEQ ID NO:3 and the peptide sequence encodedthereby are derived from EST (Expressed Sequence Tag) sequences. It isrecognised by those skilled in the art that there will inevitably besome nucleotide sequence reading errors in EST sequences (see Adams, M.D. et al, Nature 377 (supp) 3, 1995). Accordingly, the nucleotidesequence of SEQ ID NO:3 and the peptide sequence encoded therefrom aretherefore subjec to the same inherent limitations in sequence accuracy.Furthermore, the peptide sequence encoded by SEQ ID NO:3 comprises aregion of identity or close homology and/or close structural similarity(for example a conservative amino acid difference) with the closesthomologous or structurally similar protein.

Polynucleotides of the present invention may be obtained, using standardcloning and screening techniques, from a cDNA library derived from mRNAin cells of human colon tumors, placenta, and Burkitt's Lymphoma, usingthe expressed sequence tag (EST) analysis (Adams, M. D., et al. Science(1991) 252:1651-1656; Adams, M. D. et al., Nature, (1992) 355:632-634;Adams, M. D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides ofthe invention can also be obtained from natural sources such as genomicDNA libraries or can be synthesized using well known and commerciallyavailable techniques.

When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself; or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence which facilitates purification of the fusedpolypeptide can be encoded. In certain preferred embodiments of thisaspect of the invention, the marker sequence is a hexa-histidinepeptide, as provided in the pQE vector (Qiagen, Inc.) and described inGentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.The polynucleotide may also contain non-coding 5′ and 3′ sequences, suchas transcribed, non-translated sequences, splicing and polyadenylationsignals, ribosome binding sites and sequences that stabilize mRNA.

Further embodiments of the present invention include polynucleotidesencoding polypeptide variants which comprise the amino acid sequence ofSEQ ID NO:2 and in which several, for instance from 5 to 10, 1 to 5, 1to 3, 1 to 2 or 1, amino acid residues are substituted, deleted oradded, in any combination.

Polynucleotides which are identical or sufficiently identical to anucleotide sequence contained in SEQ ID NO:1, may be used ashybridization probes for cDNA and genomic DNA or as primers for anucleic acid amplification (PCR) reaction, to isolate full-length cDNAsand genomic clones encoding polypeptides of the present invention and toisolate cDNA and genomic clones of other genes (including genes encodinghomologs and orthologs from species other than human) that have a highsequence similarity to SEQ ID NO:1. Typically, these nucleotidesequences are 70% identical, preferably 80% identical, more preferably90% identical, most preferably 95% identical to that of the referent.The probes or primers will generally comprise at least 15 nucleotides,preferably, at least 30 nucleotides and may have at least 50nucleotides. Particularly preferred probes will have between 30 and 50nucleotides.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than human, may beobtained by a process which comprises the steps of screening anappropriate library under stringent hybridization conditions with alabeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof;and isolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan. Preferred stringent hybridization conditionsinclude overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1×SSC at about 65° C. Thus the present invention alsoincludes polynucleotides obtainable by screening an appropriate libraryunder stringent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or a fragment thereof.

The skilled artisan will appreciate that, in many cases, an isolatedcDNA sequence will be incomplete, in that the region coding for thepolypeptide is cut short at the 5′ end of the cDNA. This is aconsequence of reverse transcriptase, an enzyme with inherently low‘processivity’ (a measure of the ability of the enzyme to remainattached to the template during the polymerisation reaction), failing tocomplete a DNA copy of the mRNA template during 1st strand cDNAsynthesis.

There are several methods available and well known to those skilled inthe art to obtain full-length cDNAs, or extend short cDNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman et al., PNAS USA 85, 8998-9002, 1988). Recentmodifications of the technique, exemplified by the Marathon™ technology(Clontech Laboratories Inc.) for example, have significantly simplifiedthe search for longer cDNAs. In the Marathon™ technology, cDNAs havebeen prepared from mRNA extracted from a chosen tissue and an ‘adaptor’sequence ligated onto each end. Nucleic acid amplification (PCR) is thencarried out to amplify the ‘missing’ 5′ end of the cDNA using acombination of gene specific and adaptor specific oligonucleotideprimers: The PCR reaction is then repeated using ‘nested’ primers, thatis, primers designed to anneal within the amplified product (typicallyan adaptor specific primer that anneals further 3′ in the adaptorsequence and a gene specific primer that anneals further 5′ in the knowngene sequence.) The products of this reaction can then by analysed byDNA sequencing and a full-length cDNA constructed either by joining theproduct directly to the existing cDNA to give a complete sequence, orcarrying out a separate full-length PCR using the new sequenceinformation for the design of the 5′ primer.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in the art from genetically engineered host cellscomprising expression systems. Accordingly, in a further aspect, thepresent invention relates to expression systems which comprise apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., Basic Methods in Molecular Biology (1986)and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).Preferred such methods include, for instance, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector which is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate nucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook, et al., MOLECULAR CLONING,A LABORATORY MANUAL (supra). Appropriate secretion signals may beincorporated into the desired polypeptide to allow secretion of thetranslated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular environment. These signals may beendogenous to the polypeptide or they may be heterologous signals.

If a polypeptide of the present invention is to be expressed for use inscreening assays, it is generally preferred that the polypeptide beproduced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of thegene characterised by the polynucleotide of SEQ ID NO:1 which isassociated with a dysfunction will provide a diagnostic tool that canadd to, or define, a diagnosis of a disease, or susceptibility to adisease, which results from under-expression, over-expression or alteredexpression of the gene. Individuals carrying mutations in the gene maybe detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled Human Urotensin II nucleotidesequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing (ee, e.g.,Myers et al., Science (1985) 230: 1242). Sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase and S1 protection or the chemical cleavage method (see Cotton etal., Proc Natl Acad Sci USA (1985) 85: 4397-4401). In anotherembodiment, an array of oligonucleotides probes comprising HumanUrotensin II nucleotide sequence or fragments thereof can be constructedto conduct efficient screening of e.g., genetic mutations. Arraytechnology methods are well known and have general applicability and canbe used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability (seefor example: M. Chee et al., Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to the Diseases through detection of mutation in theHuman Urotensin II gene by the methods described. In addition, suchdiseases may be diagnosed by methods comprising determining from asample derived from a subject an abnormally decreased or increased levelof polypeptide or mRNA. Decreased or increased expression can bemeasured at the RNA level using any of the methods well known in the artfor the quantitation of polynucleotides, such as, for example, nucleicacid amplification, for instance PCR, RT-PCR, RNase protection, Northernblotting and other hybridization methods. Assay techniques that can beused to determine levels of a protein, such as a polypeptide of thepresent invention, in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagnostickit which comprises:

(a) a polynucleotide of the present invention, preferably the nucleotidesequence of SEQ ID NO: 1, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of the present invention, preferably the polypeptideof SEQ ID NO:2 or a fragment thereof; or

(d) an antibody to a polypeptide of the present invention, preferably tothe polypeptide of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or suspectability to a disease, particularly acuteheart failure; hypotension; hypertension; angina pectoris; myocardialinfarction; ulcers; psychotic and nuerological disorders, includinganxiety, schizophrenia, manic depression, delirium, dementia, severemental retardation and dyskinesias, amongst others.

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to, andcan hybridize with, a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound in, for example, V. McKusick, Mendelian Inheritance in Man(available on-line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease. The gene of the present invention is reported to map tohuman chromosome I.

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them, can also be used as immunogens to produceantibodies immunospecific for polypeptides of the present invention. Theterm “immunospecific” means that the antibodies have substantiallygreater affinity for the polypeptides of the invention than theiraffinity for other related polypeptides in the prior art.

Antibodies generated against polypeptides of the present invention maybe obtained by administering the polypeptides or epitope-bearingfragments, analogs or cells to an animal, preferably a non-human animal,using routine protocols. For preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples include the hybridoma technique (Kohler,G. and Milstein, C., Nature (1975) 256:495-497), the trioma technique,the human B-cell hybridoma technique (Kozbor et al., Immunology Today(1983) 4:72) and the EBV-hybridoma technique (Cole et al., MONOCLONALANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as thosedescribed in U.S. Pat. No. 4,946,778, can also be adapted to producesingle chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against polypeptides of the present invention may also beemployed to treat the Diseases, amongst others.

In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with a polypeptide of the present invention, adequate to produceantibody and/or T cell immune response to protect said animal from theDiseases hereinbefore mentioned, amongst others. Yet another aspect ofthe invention relates to a method of inducing immunological response ina mammal which comprises, delivering a polypeptide of the presentinvention via a vector directing expression of the polynucleotide andcoding for the polypeptide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromdiseases.

A further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to a polypeptide of thepresent invention wherein the composition comprises a polypeptide orpolynucleotide of the present invention. The vaccine formulation mayfurther comprise a suitable carrier. Since a polypeptide may be brokendown in the stomach, it is preferably administered parenterally (forinstance, subcutaneous, intramuscular, intravenous, or intradermalinjection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials and may bestored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in water systems and other systems known inthe art. The dosage will depend on the specific activity of the vaccineand can be readily determined by routine experimentation.

Polypeptides of the present invention are responsible for manybiological functions, including many disease states, in particular theDiseases hereinbefore mentioned. It is therefore desirous to devisescreening methods to identify compounds which stimulate or which inhibitthe function of the polypeptide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those which stimulate or which inhibit the function of thepolypeptide. In general, agonists or antagonists may be employed fortherapeutic and prophylactic purposes for such Diseases as hereinbeforementioned. Compounds may be identified from a variety of sources, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. Such agonists, antagonists or inhibitors so-identifiedmay be natural or modified substrates, ligands, receptors, enzymes,etc., as the case may be, of the polypeptide, or may be structural orfunctional mimetics thereof (see Coligan et al., Current Protocols inImmunology 1(2):Chapter 5 (1991)).

The screening method may simply measure the binding of a candidatecompound to the polypeptide, or to cells or membranes bearing thepolypeptide, or a fusion protein thereof by means of a label directly orindirectly associated with the candidate compound. Alternatively, thescreening method may involve competition with a labeled competitor.Further, these screening methods may test whether the candidate compoundresults in a signal generated by activation or inhibition of thepolypeptide, using detection systems appropriate to the cells bearingthe polypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptides may be employed in screening methods for inverseagonists or inhibitors, in the absence of an agonist or inhibitor, bytesting whether the candidate compound results in inhibition ofactivation of the polypeptide. Further, the screening methods may simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide of the present invention, to form a mixture,measuring Human Urotensin II activity in the mixture, and comparing theHuman Urotensin II activity of the mixture to a standard. Fusionproteins, such as those made from Fc portion and Human Urotensin IIpolypeptide, as hereinbefore described, can also be used forhigh-throughput screening assays to identify antagonists for thepolypeptide of the present invention (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johnson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies to the polypeptide ofthe present invention may also be used to configure screening methodsfor detecting the effect of added compounds on the production of mRNAand polypeptide in cells. For example, an ELISA assay may be constructedfor measuring secreted or cell associated levels of polypeptide usingmonoclonal and polyclonal antibodies by standard methods known in theart. This can be used to discover agents which may inhibit or enhancethe production of polypeptide (also called antagonist or agonist,respectively) from suitably manipulated cells or tissues.

The polypeptide may be used to identify membrane bound or solublereceptors, if any, through standard receptor binding techniques known inthe art. These include, but are not limited to, ligand binding andcrosslinking assays in which the polypeptide is labeled with aradioactive isotope (for instance, ¹²⁵I), chemically modified (forinstance, biotinylated), or fused to a peptide sequence suitable fordetection or purification, and incubated with a source of the putativereceptor (cells, cell membranes, cell supernatants, tissue extracts,bodily fluids). Other methods include biophysical techniques such assurface plasmon resonance and spectroscopy. These screening methods mayalso be used to identify agonists and antagonists of the polypeptidewhich compete with the binding of the polypeptide to its receptors, ifany. Standard methods for conducting such assays are well understood inthe art.

Examples of potential polypeptide antagonists include antibodies or, insome cases, oligonucleotides or proteins which are closely related tothe ligands, substrates, receptors, enzymes, etc., as the case may be,of the polypeptide, e.g., a fragment of the ligands, substrates,receptors, enzymes, etc; or small molecules which bind to the polypetideof the present invention but do not elicit a response, so that theactivity of the polypeptide is prevented.

Thus, in another aspect, the present invention relates to a screeningkit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for polypeptides of the present invention; orcompounds which decrease or enhance the production of such polypeptides,which comprises:

(a) a polypeptide of the present invention;

(b) a recombinant cell expressing a polypeptide of the presentinvention;

(c) a cell membrane expressing a polypeptide of the present invention;or

(d) antibody to a polypeptide of the present invention;

which polypeptide is preferably that of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

It will readily appreciated by the skilled artisan that a polypeptide ofthe present invention may also be used in a method for thestructure-based design of an agonist, antagonist or inhibitor of thepolypeptide, by:

(a) determining in the first instance the three-dimensional structure ofthe polypeptide;

(b) deducing the three-dimensional structure for the likely reactive orbinding site(s) of an agonist, antagonist or inhibitor;

(c) synthesing candidate compounds that are predicted to bind to orreact with the deduced binding or reactive site; and

(d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

It will be further appreciated that this will normally be an interativeprocess.

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, acute heart failure;hypotension; hypertension; angina pectoris; myocardial infarction;ulcers; psychotic and neurological disorders, including anxiety,schizophrenia, manic depression, delirium, dementia, severe mentalretardation and dyskinesias, related to either an excess of, or anunder-expression of, Human Urotensin II polypeptide activity.

If the activity of the polypeptide is in excess, several approaches areavailable. One approach comprises administering to a subject in needthereof an inhibitor compound (antagonist) as hereinabove described,optionally in combination with a pharmaceutically acceptable carrier, inan amount effective to inhibit the function of the polypeptide, such as,for example, by blocking the binding of ligands, substrates, receptors,enzymes, etc., or by inhibiting a second signal, and thereby alleviatingthe abnormal condition. In another approach, soluble forms of thepolypeptides still capable of binding the ligand, substrate, enzymes,receptors, etc. in competition with endogenous polypeptide may beadministered. Typical examples of such competitors include fragments ofthe Human Urotensin II polypeptide.

In still another approach, expression of the gene encoding endogenousHuman Urotensin II polypeptide can be inhibited using expressionblocking techniques. Known such techniques involve the use of antisensesequences, either internally generated or separately administered (see,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression of HumanUrotensin II and its activity, several approaches are also available.One approach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates a polypeptide of thepresent invention, i.e., an agonist as described above, in combinationwith a pharmaceutically acceptable carrier, to thereby alleviate theabnormal condition. Alternatively, gene therapy may be employed toeffect the endogenous production of Human Urotensin II by the relevantcells in the subject. For example, a polynucleotide of the invention maybe engineered for expression in a replication defective retroviralvector, as discussed above. The retroviral expression construct may thenbe isolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing RNA encoding a polypeptide of thepresent invention such that the packaging cell now produces infectiousviral particles containing the gene of interest. These producer cellsmay be administered to a subject for engineering cells in vivo andexpression of the polypeptide in vivo. For an overview of gene therapy,see Chapter 20, Gene Therapy and other Molecular Genetic-basedTherapeutic Approaches, (and references cited therein) in HumanMolecular Genetics, T Strachan and A P Read, BIOS Scientific PublishersLtd (1996). Another approach is to administer a therapeutic amount of apolypeptide of the present invention in combination with a suitablepharmaceutical carrier.

In a further aspect, the present invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of apolypeptide, such as the soluble form of a polypeptide of the presentinvention, agonist/antagonist peptide or small molecule compound, incombination with a pharmaceutically acceptable carrier or excipient.Such carriers include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theinvention further relates to pharmaceutical packs and kits comprisingone or more containers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides and othercompounds of the present invention may be employed alone or inconjunction with other compounds, such as therapeutic compounds.

The composition will be adapted to the route of administration, forinstance by a systematic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

The dosage range required depends on the choice of peptide or othercompounds of the present invention, the route of administration, thenature of the formulation, the nature of the subject's condition, andthe judgment of the attending practitioner. Suitable dosages, however,are in the range of 0.1-100 μg/kg of subject. Wide variations in theneeded dosage, however, are to be expected in view of the variety ofcompounds available and the differing efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by intravenous injection.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as “gene therapy” asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

Polynucleotide and polypeptide sequences from a valuable informationresource with which to identify further sequences of similar homology.This is most easily facilitated by storing the sequence in a computerreadable medium and then using the stored data to search a sequencedatabase using well known searching tools, such as GCC. Accordingly, ina further aspect, the present invention provides for a computer readablemedium having stored thereon a polynucleotide comprising the sequence ofSEQ ID NO:1 and/or a polypeptide sequence encoded thereby.

The following definitions are provided to facilitate understanding ofcertain terms used frequently hereinbefore.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immumoglobulinexpression library.

“Isolated” means altered “by the hand of man” from the natural state. Ifan “isolated” composition of substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not “isolated,” but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is “isolated”, as theterm is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polynucleotide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalant attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination (see, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993; Wold, F., Post-translational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Post-translational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62).

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published technique (see, e.g.:COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford UniversityPress, New York, 1998; BIOCOMPUTING INFORMATICS AND GENOME PROJECTS,Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OFSEQUENCE DATA, PART 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje. G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCG program package (Devereux, J., et al., Nucleic AcidsResearch (1984) 12(1):387), BLASTP, BLASTN, and FASTA (Atschul, S. F. etal., J Molec Biol (1990) 215:403).

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO:1, that is be100% identical, or it may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence. Suchalterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleotide alterations is determined by multiplying the total number ofnucleotides in SEQ ID NO:1 by the numerical percent of the respectivepercent identity(divided by 100) and subtracting that product from saidtotal number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, and y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integerproduct of x_(n) and y is rounded down to the nearest integer prior tosubtracting it from x_(n). Alterations of a polynucleotide sequenceencoding the polypeptide of SEQ ID NO:2 may create nonsense, missense orframeshift mutations in this coding sequence and thereby alter thepolypeptide encoded by the polynucleotide following such alterations.

Similarly, a polypeptide sequence of the present invention may beidentical to the reference sequence of SEQ ID NO:2, that is be 100%identical, or it may include up to a certain integer number of aminoacid alterations as compared to the reference sequence such that the %identity is less than 100%. Such alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of amino acidalterations for a given % identity is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the numerical percent ofthe respective percent identity(divided by 100) and then subtractingthat product from said total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, and y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integerproduct of x_(a) and y is rounded down to the nearest integer prior tosubtracting it from x_(a).

“Fusion protein” refers to a protein encoded by two, often unrelated,fused genes or fragments thereof. In one example, EP-A-0 464 disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [seee.g., EP-A 0232 262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

SEQUENCE INFORMATIONTGCCGGTGCCTTCATGGGCAGGACTAGGCCGAAAACTCCCCAGATTGTCATTCTTCAGGG SEQ ID NO:1 ATGGCAGCCCTAAACACAGCATGGCAACTCATCTACTCACTCATGAAAGATTAAAAAATGGAAACCAACGTATTTCATCTTATGCTCTGCGTCACTTCTGCTCGGACTCATAAATCCACGTCTCTTTGCTTTGGCCACTTCAACTCATATCCAAGCCTTCCTTTAATTCATGATTTATTGCTGGAAATATCCTTTCAACTCTCAGCACCTCATGAAGACGCGCGCTTAACTCCGGAGGAGCTAGAAAGAGCTTCCCTTCTACAGATACTGCCAGAGATGCTGGGTGCAGAAAGAGGGGATATTCTCAGGAAAGCAGACTCAAGTACCAACATTTTTAACCCAAGAGGAAATTTGAGAAAGTTTCAGGATTTCTCTGGACAAGATCCTAACATTTTACTGAGTCATCTTTTGGCCAGAATCTGGAAACCATACAAGAAACGTGAGACTCCTGATTGCTTCTGGAAATACTGTGTCTGAAGTGAAATAAGCATCTGTTAGTCAGCTCAGAAACACCCATCTTAGAATATGAAAAATAACACAATGCTTGATTTGAAAACAGTGTGGAGAAAAACTAGGCAAACTACACCCTGTTCATTGTTACCTGGAAAATAAATCCTCTATGTTTTGCMETNVFHLMLCVTSARTHKSTSLCFGHFNSYPSLPLIHDLLLEISFQLSAPHEDARLTPE SEQ ID NO:2ELERASLLQILPEMLGAERGDILRKADSSTNIFNPRGNLRKFQDFSGQDPNILLSHLLARIWKPYKKRETPDCFWKYCVTGAAGGATGAACCCAATTCTAAGTATCTGTTTTTACTCTGTAGCACCTCATGAAGACGCG SEQ ID NO:3CGCTTAACTCCGGAGGAGCTAGAAAGAGCTTCCCTTCTACAGATACTGCCAGAGATGCTGGGTGCAGAAAGAGGGGATATTCTCAGGAAAGCAGACTCAAGTACCAACATTTTTAACCCAAGAGGAAATTTGAGAAAGTTTCAGGATTTCTCTGGACAAGATCCTAACATTTTACTGAGTCATCTTTTGGCCAGAATCTGGAAACCATACAAGAAACGTGAGACTCCTGATTGCTTCTGGAAATACTGTGTCTGARMNPILSICFYSVAPHEDARLTPEELERASLLQILPEMLGAERGDILRKADSSTNIFNP SEQ ID NO:4RGNLRKFQDFSGDPNILLSHLLARIWKPYKKRETPDCFWKYCV ETPDCFWKYCV SEQ ID NQ:5

5 1 688 DNA HOMO SAPIENS 1 tgccggtgcc ttcatgggca ggactaggcc gaaaactccccagattgtca ttcttcaggg 60 atggcagccc taaacacagc atggcaactc atctactcactcatgaaaga ttaaaaaatg 120 gaaaccaacg tatttcatct tatgctctgc gtcacttctgctcggactca taaatccacg 180 tctctttgct ttggccactt caactcatat ccaagccttcctttaattca tgatttattg 240 ctggaaatat cctttcaact ctcagcacct catgaagacgcgcgcttaac tccggaggag 300 ctagaaagag cttcccttct acagatactg ccagagatgctgggtgcaga aagaggggat 360 attctcagga aagcagactc aagtaccaac atttttaacccaagaggaaa tttgagaaag 420 tttcaggatt tctctggaca agatcctaac attttactgagtcatctttt ggccagaatc 480 tggaaaccat acaagaaacg tgagactcct gattgcttctggaaatactg tgtctgaagt 540 gaaataagca tctgttagtc agctcagaaa cacccatcttagaatatgaa aaataacaca 600 atgcttgatt tgaaaacagt gtggagaaaa actaggcaaactacaccctg ttcattgtta 660 cctggaaaat aaatcctcta tgttttgc 688 2 139 PRTHOMO SAPIENS 2 Met Glu Thr Asn Val Phe His Leu Met Leu Cys Val Thr SerAla Arg 1 5 10 15 Thr His Lys Ser Thr Ser Leu Cys Phe Gly His Phe AsnSer Tyr Pro 20 25 30 Ser Leu Pro Leu Ile His Asp Leu Leu Leu Glu Ile SerPhe Gln Leu 35 40 45 Ser Ala Pro His Glu Asp Ala Arg Leu Thr Pro Glu GluLeu Glu Arg 50 55 60 Ala Ser Leu Leu Gln Ile Leu Pro Glu Met Leu Gly AlaGlu Arg Gly 65 70 75 80 Asp Ile Leu Arg Lys Ala Asp Ser Ser Thr Asn IlePhe Asn Pro Arg 85 90 95 Gly Asn Leu Arg Lys Phe Gln Asp Phe Ser Gly GlnAsp Pro Asn Ile 100 105 110 Leu Leu Ser His Leu Leu Ala Arg Ile Trp LysPro Tyr Lys Lys Arg 115 120 125 Glu Thr Pro Asp Cys Phe Trp Lys Tyr CysVal 130 135 3 315 DNA HOMO SAPIENS 3 tgaaggatga acccaattct aagtatctgtttttactctg tagcacctca tgaagacgcg 60 cgcttaactc cggaggagct agaaagagcttcccttctac agatactgcc agagatgctg 120 ggtgcagaaa gaggggatat tctcaggaaagcagactcaa gtaccaacat ttttaaccca 180 agaggaaatt tgagaaagtt tcaggatttctctggacaag atcctaacat tttactgagt 240 catcttttgg ccagaatctg gaaaccatacaagaaacgtg agactcctga ttgcttctgg 300 aaatactgtg tctga 315 4 103 PRT HOMOSAPIENS 4 Arg Met Asn Pro Ile Leu Ser Ile Cys Phe Tyr Ser Val Ala ProHis 1 5 10 15 Glu Asp Ala Arg Leu Thr Pro Glu Glu Leu Glu Arg Ala SerLeu Leu 20 25 30 Gln Ile Leu Pro Glu Met Leu Gly Ala Glu Arg Gly Asp IleLeu Arg 35 40 45 Lys Ala Asp Ser Ser Thr Asn Ile Phe Asn Pro Arg Gly AsnLeu Arg 50 55 60 Lys Phe Gln Asp Phe Ser Gly Gln Asp Pro Asn Ile Leu LeuSer His 65 70 75 80 Leu Leu Ala Arg Ile Trp Lys Pro Tyr Lys Lys Arg GluThr Pro Asp 85 90 95 Cys Phe Trp Lys Tyr Cys Val 100 5 11 PRT HOMOSAPIENS 5 Glu Thr Pro Asp Cys Phe Trp Lys Tyr Cys Val 1 5 10

What is claimed is:
 1. An isolated polynucleotide encoding thepolypeptide consisting of the amino acid sequence set forth in SEQ IDNO:
 5. 2. An isolated polynucleotide which is fully complementary to thepolynucleotide of claim
 1. 3. An isolated polynucleotide encoding afusion polypeptide, said fusion polypeptide consisting of the amino acidsequence of SEQ ID NO: 5 fused to a marker sequence to facilitatepurification of said fusion polypeptide.
 4. An isolated polynucleotideencoding a fusion polypeptide, said fusion polypeptide consisting of aleader or secretory sequence and the amino acid sequence of SEQ ID NO:5.